Culturing apparatus

ABSTRACT

A culturing apparatus has a culture vessel having a first elastic seal bonded on its upper surface and a culture space formed thereon, and a joint for supplying solution such as a medium to the culture vessel and a second elastic seal having microprojection formed at the lower face. The first elastic seal has a valve for supplying or discharging solution. The second elastic seal is formed with microprojection at the position corresponding to a valve for preventing a spill. The culture vessel and the joint are sucked between the first elastic seal and the second elastic seal, thereby forming an integral seeding device.

CLAIM OF PRIORITY

The present application claims priority from Japanese application serialJP2004-341399 filed on Nov. 26, 2004, the content of which is herebyincorporated by reference into this application.

BACKGROUND OF THE INVENTION

The present invention relates to a culturing apparatus that culturescells.

Japanese Patent Laid-Open No. 2002-262856 discloses an example of aconventional automated cell culture. In a cell culturing apparatusdisclosed in this application, an automatic medium exchanging device isprovided with an incubator, a medium exchanging robot and a managementcomputer, in order to automatically exchange a medium. The managementcomputer takes out a culture tray, on which cells are cultured, from theincubator to the outside by a carrier robot or the like, discharges aliquid medium in the culture tray with a needle, injects new liquidmedium by the needle, and puts the culture tray into the incubator.

Japanese Patent Laid-Open No. 2004-129568 discloses an example of aculture vessel, in a cell culture, that reduces the infection andachieves the effective cell culture with a simple configuration. Theculture vessel disclosed in this application has a vessel body whoseinside is sterilized and that has a film disposed at its portion and atleast one tube that communicates with the inside of this vessel body andis made of a thermally depositable material whose leading end is closed.

The most important subject upon culturing cells are to prevent thecultured cell from being infected by dust, bacteria, virus, or the like.In order to prevent the infection, it is required that a series ofprocesses in the cell culture such as cell seeding or exchange of amedium is performed in a sterilized state so as to prevent contaminantsfrom being entered. A cell culture or processing is currently made byskilled technicians in a cell processing center at a large-scale culturefacility composed of sterilized each facility by using a techniqueprovided by GMP (GOOD MANUFACTURING PRACTICE). In this infectionmanagement, it is difficult to enhance productivity and reduce cost forindustrialization.

In order to eliminate this inconvenience, an automated culturingapparatus disclosed in Japanese Patent Laid-Open No. 2002-262856 hasbeen proposed wherein an operation same in level as that carried out bya skilled technician with a quality retained is possible by using anautomated technique. The device disclosed in this application performscells seeding or exchange of a medium by using a driving device such asa robot arm, so that it requires a clean space or control systemconsidering an operating range of the arm, thereby making the wholedevice extremely large-scale.

Since a current culture tool such as a culture dish or the like is opento the outer space, safety cannot be assured so long as germs of varioussorts in the space around a seeding device are reduced. Therefore, theautomated culturing apparatus can only be installed in a facility, thewhole of which is sterilized, such as a processing center, so that alarge sterilizing system is required to produce a small tissue fromcultured cells.

The infection can be prevented by closing the culture space to removethe connection with the outside in order to eliminate suchinconvenience, as disclosed in JP2004-129568. A device can beminiaturized by using the culture vessel disclosed in this application,but a perfect closed system makes it difficult to move substances intoor from an outer space.

BRIEF SUMMARY OF THE INVENTION

The present invention is accomplished in view of the aforesaidinconvenience of the conventional technique, and its object is to simplyperform the cell seeding and exchange of medium, while preventing theinfection from the outside in the cell culture.

The feature of the invention for accomplishing the aforesaid object isto comprise a culture vessel having a culture vessel member to which aculture space for culturing cells are formed and a first elastic sealarranged above the culture space of the culture vessel member, and ajoint having a joint member provided with supplying means for supplyingsolution such as a medium to this culture vessel and a second elasticseal bonded to the joint member, wherein the culture vessel isdetachably mounted to the joint by using suction means formed on thejoint.

In this feature, the suction means has a suction pad attached to thesecond elastic seal and a pump that communicates with this suction padto exert suction force, and the first elastic seal has cutout at pluralsections, this cutout being used as a valve. Further, holes are formedon the second elastic seal at the positions corresponding to cutouts,wherein a ring-like step portion having a predetermined thickness isformed around each hole, whereby the first elastic seal is pushed topenetrate through the second elastic seal, to attach a tube, which cansupply the solution into the culture space, to the joint member.

Another feature of the invention for accomplishing the aforesaid objectis a culturing apparatus comprising a culture vessel having a firstelastic seal bonded to its upper face and having the culture spaceformed therein; and a joint provided with supplying section forsupplying solution such as a medium to the culture vessel and havingbonded to its lower face a second elastic seal having a microprojection;wherein the first elastic seal has a valve for supplying or dischargingthe solution, and second elastic seal has a microprojection formed atthe position corresponding to the valve for preventing a spill, whereinthe culture vessel and the joint are sucked between the first elasticseal and the second elastic seal to compose an integrally formed seedingdevice.

In this feature, adsorbing means is provided at the second elastic sealand a suction pump is connected to this adsorbing means via a tube,wherein the culture vessel and the joint are attached to and detachedfrom each other between the first elastic seal and the second elasticseal by the operation of the suction pump. It is preferable that awater-repellant process is provided on the surface of the valve and thesurface of the microprojection. Further, it is desirable that a base onwhich the seeding device is placed is provided, wherein a projection isformed on the base at the position corresponding to the width of theculture vessel, a groove is provided at the side section of the culturevessel into which the projection is engaged, and the seeding device canbe held by engaging the projection into the groove upon the culture.

In the aforesaid feature, an arm may be provided and a rotation axis isconnected to the leading end of this arm, wherein, when solution such asa medium is injected into or discharged from the culture space, therotation axis may be operated such that the seeding device integrallyformed by the adsorbing means is inclined from its horizontal direction.It is preferable that plural valves are formed at the positioncorresponding to the outside of the culture space, wherein the solutionis supplied from the valve section positioned downward and the solutionis discharged from the valve section positioned upward, when thesolution is exchanged.

According to the present invention, a detachable joint is provided at aseeding device, and this joint is sealed by a resin, whereby an exchangeof a medium or cell seeding can be easily performed while preventing theinfection from the outside. Further, liquid is injected from below withthe attached joint and seeding device inclined in the verticaldirection, whereby the liquid is assuredly injected into the culturespace. The present invention also provides an effect that bubblesincluded in the culture space are vented at this time.

BRIEF DESCRIPTION OF THE SEVERAL VIEW OF THE DRAWING

FIGS. 1 to 5 are views showing one embodiment of a seeding deviceaccording to the present invention, wherein FIG. 1 is its perspectiveview:;

FIG. 2 is its exploded perspective view;

FIG. 3 is a partial detailed sectional view;

FIG. 4 is its front view showing a state of removing a culture vessel;

FIG. 5 is a front view when the seeding device is inclined; and

FIG. 6 is a top plan view of a culturing apparatus according to oneembodiment of the present invention; and

FIG. 7 is a perspective view showing a joint holder positioning sectionused for the culturing apparatus.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of a culturing apparatus according to the invention willbe explained hereinafter with reference to drawings. FIG. 6 is a topplan view of a culturing apparatus 110. The culturing apparatus 110 hasa culture vessel inserting portion 112 for inserting a culture vessel 60into the sterilized culturing apparatus 110 and a culture chamber 113for culturing cells in the culturing vessel 60. The culture vessel 60inserted from the culture vessel inserting portion 112 into theculturing apparatus 110 is transported to the culture chamber 113 bymoving one of a pair of joint holder positioning sections 111, that arearranged in the culturing apparatus 110 so as to be substantiallyparallel to each other, in the forward and rearward directions 111 a and111 b.

A turntable 96 having a driving system provided at its back face isarranged in the culture chamber 113. Plural bases 95 are spaced in thecircumferential direction on the turntable 96. When cells are put intothe culture vessel 60 at the beginning of the culture, the other one ofthe joint holder positioning sections 111 moves in the forward andrearward directions 111 a and 111 b, so that cell suspension put from acell inserting section 114 provided at the culturing apparatus 110 isinjected into the culture vessel 60. The culture vessel 60 to which thecell suspension is injected is returned to the culture chamber 113,whereby the culture is progressing. When a medium is exchanged, a mediumsupplied from a medium supplying section 115 is injected into theculture vessel 60 held by the joint holder positioning sections 111. Themedium supplying section 115 is placed at the outside of a sterilizeddraft 116. When the culture is completed, the joint holder positioningsections 111 returns the culture vessel to the culture vessel insertingsection 112 or a culture vessel removing section separately provided.

The state of a cell seeding or exchange of a medium will be explainedwith reference to FIGS. 1 to 5. FIG. 1 is a perspective view showing astate in which a seeding device 70 having the culture vessel 60 and ajoint 50 integrally formed is attached on the turntable 96. FIG. 2 is anexploded perspective view of the seeding device 70 shown in FIG. 1. FIG.3 is a partial detailed sectional view of the seeding device 70. Theseeding device 70 has the culture vessel 60 having a vessel member 40 towhich a culture space 42 b is formed at its center, and the joint 50that is arranged on the culture vessel 60 for introducing or dischargingthe culture liquid into or from the culture space 42 b.

The vessel member 40 has a shape of thin rectangular parallel-epiped,and has the circular hollow culture space 42 b formed at its center. Afilm 42 a is attached to the bottom face of the vessel member 40 with anadhesion. Four locking holes 41 are formed around the circular space ofthe vessel member 40. Formed in the vicinity of the bottom face of thelocking holes 41 is a flow channel 43 for communicating the culturespace 42 b with the locking holes 41.

A rectangular first elastic seal 30 having valves 31 formed at theposition corresponding to each locking hole 41 is held at the upper faceof the vessel member 40 with an adhesion. The first elastic seal is madeof a viscoelastic material such as a silicon resin, wherein each of thevalves 31 is a cross cutout formed at this viscoelastic material. Thevessel member 40 and the first elastic seal 30 compose the culturevessel 60.

The joint 50 has a rectangular second elastic seal 20 arranged at theupper face of the culture vessel 60 and a joint member 10 that is madeby fixing the second elastic seal 20 with an adhesion to be formed intoa thin rectangular parallel-epiped. The second elastic seal 20 is madeof a viscoelastic material such as a silicon resin. The second elasticseal 20 has annular microprojections 21, each being formed at theposition opposite to the first elastic seal 30 and corresponding to eachof the locking holes 41 formed at the vessel member 40. Holes 22 for asuction pad whose detail will be explained later are formed at thecorner sections of the second elastic seal 20.

The joint member 10 is formed into a thin rectangular parallel-epipedand has four corner sections. Attached to the bottom face thereof arehorn-like adsorbing pads 15 at the position corresponding to each of theholes 22 for a suction pad formed on the second elastic seal 20.Through-holes to which tubes 13 fitted to the locking holes 41 on thevessel member 40 are attached are formed on the bottom face of the jointmember 10. As shown in FIG. 3, each through-hole is a steppedthrough-hole, wherein the upper hole is a threaded hole to which afitted connector 11 is attached.

A tube 12 that can supply cell suspension, medium or chemical isdetachably connected to the connector 11. Holes that communicate withthe back face of the adsorbing pads 15 are also formed on the jointmember 10. A suction tube 14 is fitted to each hole. Further, an arm 80is attached to one side face of the joint member 10.

The joint 50 and the culture vessel 60 are made integral by a suctionfrom the suction tubes 14, thereby composing the seeding device 70. Theintegrated seeding device 70 is fixed on the base 95 by an engagementbetween projections 91 formed on the base 95 and grooves 90 formed atthe periphery section at the bottom section of the culture vessel 60.

A culture method by using the seeding device 70 having the aforesaidconfiguration will be explained hereinafter. Firstly, the state ofadsorbing the culture vessel 60 to the joint 50 will be explained byusing FIG. 3. Holes 41 for tubes 13 are formed on the culture vesselmember 40, so that, if the culture vessel member 40 is handled as it isopened, it may be infected by germs or the like from the outside.Further, there is a fear that the environment may be polluted by aliquid spill from the holes 41. New tubes 13 are inserted into the holes41 formed on the vessel member 40, every time a medium is exchanged, inorder to prevent the infection. However, this increases the exchangefrequency of tubes 13. Accordingly, it is necessary that the tubes 13and holes 41 are assuredly sealed, even if the exchange frequency isincreased, and that the holes 41 are automatically closed so as not tocause a liquid spill from the holes 41 even if the tubes 13 are removed.In view of this, the following seal is used in order to prevent that theinside of the culture vessel 60 is infected.

Specifically, in order to enhance adhesiveness and liquid spillprevention performance upon attaching or detaching the culture vessel 60and the joint 50, the second elastic seal 20 is adhered onto the bottomface of the joint member 10 and the first elastic seal 30 is adheredonto the upper face of the culture vessel member 40. Then, the firstelastic seal 30 is brought into intimate contact with the second elasticseal 20 by using a suction pump. A viscoelastic material is used forthese seals 20 and 30 for enhancing adhesiveness between the secondelastic seal 20 and the first elastic seal 30.

A valve 31 composed of a slit 31 a is formed on the first elastic seal30 for preventing the inclusion of germs or liquid spill from the holes41, and for serving as a cover that automatically closes the hole 41when the tube 13 is not inserted into the hole 41. A seal member 31 bthat is pushed downward from the slit 31 a upon the insertion of thetube 13 is recovered by elastic force when the tube 13 is not insertedinto the hole 41, whereby the valve 31 keeps its closed state. As aresult, a liquid spill can be prevented.

When the tube 13 is inserted into the slit for the culture, the sealmember 31 b is downwardly spread out, so that the valve 31 is opened.The seal member 31 b composing the valve 31 is brought into intimatecontact with the tube 13 with elastic force, so that the liquid spilland reversed flow are prevented. It should be noted that water-repellantprocess is provided for the valve 31 for preventing the liquid spill andinfection.

On the other hand, annular microprojections 21 are formed around theholes for the tubes 13 of the second elastic seal 20. They are used foradsorption between the first elastic seal 30 and the second elastic seal20. When the joint 50 and the culture vessel 60 are adsorbed to eachother, vacuum adsorption is performed by using the adsorbing pad 15shown in detail in FIG. 4. The adsorbing pad 15 is connected to asuction pump not shown via the suction tube 14. Upon the suction, themicroprojection 21 is pushed toward the culture vessel 60 to enhance theadhesiveness at the seal section formed around the hole 41.

Since the microprojections 21 are formed on the second elastic seal 20,the contact area between the first elastic seal 30 and the secondelastic seal 20 is reduced, thereby increasing the contact pressure atthe contact section. On the other hand, if the microprojections 21 arenot formed, the contact pressure between the first elastic seal 30 andthe second elastic seal 20 is reduced, thereby deteriorating the sealingperformance around the tube 13.

Further, providing the microprojections 21 facilitates the removal ofthe culture vessel 60 from the joint 50. The first and second elasticseal members 30 and 20 have excellent adhesiveness since they are madeof viscoelastic material. The step of each of the microprojection 21prevents that the sections other than the seal section, i.e., theannular microprojections 21, are unnecessarily brought into intimatecontact with the culture vessel 60. The formation of themicroprojections 21 prevents the liquid spill between the joint 50 andthe culture vessel 60, and further prevents that the liquid remains inthe vicinity of the tube 13 to pollute the surroundings when the joint50 is removed. Moreover, the water-repellant process is provided on themicroprojections 21, so that there is no chance that the medium adheresto the microprojections 21.

Subsequently, a method for injecting the cell suspension or medium intothe culture vessel 60 will be explained with reference to FIGS. 4, 5 and7. FIG. 4 is a front view of the seeding device 70. The joint 50 isarranged above the horizontally placed culture vessel 60. The arm 80 isattached at the side section of the joint 50, wherein the end section ofthe arm 80 is connected to a rotation axis 81. The rotation axis 81 canbe moved in the upward direction 70 a and downward direction 70 b.

Upon positioning the joint 50, the tube 13 attached to the joint 50 ismatched to the hole 41 formed on the culture vessel 60. As shown in FIG.3, the tube 13 fixed to the joint 50 pushes the seal member 31 bdownward, so that the tube 13 is inserted into the hole 41 formed on theculture vessel 60. After the joint 50 is placed on the culture vessel60, a suction pump not shown is driven, whereby the adsorbing pad 15 towhich the suction tube 14 is connected sucks the culture vessel 60 andbrings the same into intimate contact with the joint 50. Thisestablishes an intimate contact between the second resin seal 20 and thefirst resin seal 30.

When the joint 50 and the culture vessel 60 are adhered with each otherto compose the integral seeding device 70, the integral seeding device70 is inclined by using the rotation axis 81. FIG. 5 is a front view ofthe seeding device 70. The rotation axis 81 is rotated 90 degrees in aclockwise direction 81 a. The adhered culture vessel 60 and the joint 50are rotated from the horizontal direction to the vertical directionshown by an arrow of 70 c.

With this state, the medium or cell suspension is injected from the tube12 positioned in the downward direction in the culture space 42 b, i.e.,from the direction shown by an arrow 12 b. On the other hand, wasteliquid or gas in the seeding device 70 is discharged from the tube 12positioned in the upward direction, i.e., from the direction shown by anarrow 12 a. The injected medium is the one kept in cold storage atanother place. This medium is warmed up to a suitable temperatureimmediately before the use, and supplied to the culture space 42 b witha pump not shown as afresh medium. The liquid injecting operation andliquid discharging operation are performed with the seeding device 70standing in the vertical direction, whereby the bubbles intruded in theculture space 42 b can be vented.

After a predetermined liquid is injected, the rotation axis 81 isrotated in a counterclockwise direction 81 b. The integrated seedingdevice 70 is returned in the horizontal direction from the verticaldirection. The rotation axis 81 movable in the upward and downwarddirections is operated, thereby setting the seeding device 70 on thebase 95 shown in FIG. 1. Projections 91 that are engaged with grooves 90are formed at the side face of the seeding device 70. Therefore, theseeding device 70 is positioned at the predetermined location before theengagement. Thereafter, the seeding device 70 is moved in the horizontaldirection 92 a to engage the projections 91 into the grooves 90.

The suction pump is stopped for stopping the suction from the adsorbingpad 15. Thereafter, pressure air is supplied to the adsorbing pad 15.When the pressure air is blown, resilience is caused instead of thesuction force between the culture vessel 60 and the joint 50, so thatthe adhesion between the culture vessel 60 and the joint 50 is lost.Since the adhesion between the culture vessel 60 and the joint 50 islost, the rotation axis 80 movable in the upward and downward directions70 a and 70 b is operated in the upward direction 70 a to separate theculture vessel 60 from the joint 50.

When the culture vessel 60 is lifted up in the direction of 70 a, therotation axis 80 is operated in the downward direction 70 b in orderthat the joint 50 and the culture vessel 60 are adsorbed and adhered toeach other. Thereafter, the seeding device 70 is moved in the direction92 b in which the engagement between the projections 91 and the grooves90 is released as shown in FIG. 1. When the engagement between theprojections 91 and the grooves 90 is released, the seeding device 70 ismoved in the upward direction 70 a, and the liquid is injected. Thisseries of operation procedure can keep the culture space 42 b in theclosed state as much as possible, even if the cell suspension or mediumis put into the culture vessel 60. Thus, the spill or infection from theculture space 42 b can be prevented.

FIG. 7 is a perspective view showing a joint holder positioning section111 used in this series of operation. A rail 121 is attached along themoving direction such that a stand 120 is movable in the forward andbackward directions 111 a and 111 b. The stand 120 is fitted to the railat its base section. A gear shaft 123 movable in the upward and downwarddirections 70 a and 70 b is attached to a shaft 122 of the stand 120extending in the vertical direction. A rack 124 is meshed with the gearshaft 123, whereby the rack 124 advances or retreats in the leftward andrightward directions 92 a and 92 b when the gear shaft 123 is rotated.The rotation axis 81 is attached horizontally at the leading end of therack 124 so as to be parallel to the rail. The arm 80 of the seedingdevice 70 can be attached to the rotation axis 80. When the rotationaxis 81 is rotated, the seeding device 70 is pivoted in the directionsof 81 a and 81 b about the rotation axis 80 extending in the frontwardand backward directions 111 a and 111 b.

1. A culturing apparatus comprising: a culture vessel having a firstelastic seal bonded to its upper face and having a culture space formedtherein; and a joint provided with a supplying section for supplyingsolution as a medium to the culture vessel and a suction pad for pullingthe culture vessel, and having bonded to its lower face a second elasticseal having a microprojection; wherein the first elastic seal has avalve for supplying or discharging the solution, wherein the secondelastic seal has the microprojection, which is integrated therein and isformed at the position corresponding to the valve for preventing aspill, wherein the culture vessel and the joint compose a seeding devicein which the first elastic seal and the second elastic seal areconnected via the microprojection by the operation of the suction pad,and wherein each of the first elastic seal and the second elastic sealcomprises an inserting section for detachably inserting a tube that isable to supply and discharge the solution.
 2. The culturing apparatusaccording to claim 1, wherein the first elastic seal has cutouts atplural sections, wherein each cutout being used as a valve.
 3. Theculturing apparatus according to claim 2, wherein the second elasticseal comprises holes at the positions corresponding to the cutouts andthe microprojection constituted by an annular microprojection that has apredetermined thickness and is formed around each of the holes, and thetube penetrates through the second elastic seal so as to be able tosupply the solution to the culture space and is detachably attached tothe joint.
 4. The culturing apparatus according to claim 1 wherein thesuction pad is provided at the second elastic seal and is connected to asuction pump via a tube, wherein the culture vessel and the joint areattached to and detached from each other between the first elastic sealand the second elastic seal by the operation of the suction pump.
 5. Theculturing apparatus according to claim 1, wherein a water-repellantprocess comprises treating the surface of the valve and themicroprojection.
 6. The culturing apparatus according to claim 1,wherein a base on which the seeding device is placed is provided,wherein a projection is formed on the base at the position correspondingto the width of the culture vessel, a groove is provided at the sidesection of the culture vessel into which the projection is engaged, andthe seeding device can be held by engaging the projection into thegroove upon the culture.
 7. The culturing apparatus according to claim1, wherein an arm is provided on a side surface of the joint and arotation axis is connected to the leading end of this arm, wherein, whenthe solution injected into or discharged from the culture space, therotation axis is operated such that the seeding device integrally formedby the suction member is inclined from the horizontal condition.
 8. Theculturing apparatus according to claim 7 wherein the valve isconstructed of plural valves formed at the outside of the culture space,wherein the solution is supplied from the valve positioned downward andthe solution is discharged from the valve positioned upward, when thesolution is exchanged.
 9. The culturing apparatus according to claim 1,wherein the first elastic seal and the second elastic seal are made ofviscoelastic material.
 10. The culturing apparatus according to claim 1wherein the microprojection is formed in a annular shape and isintegrated with the second elastic seal.
 11. The culturing apparatusaccording to claim 1 wherein a thickness of the first elastic seal issmaller than that of the second elastic seal.
 12. A culturing apparatuscomprising: a culture vessel having a first elastic seal bonded to itsupper face and having a culture space formed therein; and a jointprovided with a supplying section for supplying solution as a culturemedium to the culture vessel and a suction member for pulling theculture vessel, and having bonded to its lower face a second elasticseal having a microprojection; wherein the first elastic seal has avalve for supplying or discharging the solution, wherein the secondelastic seal has the microprojection, which is integrated therein and isformed at the position corresponding to the valve, for preventing aspill, and wherein the culture vessel and the joint compose a seedingdevice in which the first elastic seal and the second elastic seal areconnected via the microprojection by the operation of the suctionmember, each of the first elastic seal and the second elastic sealcomprises a plurality of inserting sections for detachably insertingsections for detachably inserting a plurality of tubes, an arm isprovided on a side surface of the joint, a rotation axis is connected tothe leading end of this arm, the rotation axis is operated such that theseeding device integrally formed by the suction member is moved from thehorizontal condition to the vertical condition, when the solution isinjected into or discharged from the culture space, gas or the solutionis discharged from the valve positioned upward and the solution issupplied from the valve positioned downward in the vertical condition.